Pens for biological micro-objects

ABSTRACT

Individual biological micro-objects can be deterministically selected and moved into holding pens in a micro-fluidic device. A flow of a first liquid medium can be provided to the pens. Physical pens can be structured to impede a direct flow of the first medium into a second medium in the pens while allowing diffusive mixing of the first medium and the second medium. Virtual pens can allow a common flow of medium to multiple ones of the pens.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a non-provisional (and thus claims the benefit) ofU.S. provisional patent application Ser. No. 61/720,956 (filed Oct. 31,2012), which is incorporated by reference herein in its entirety.

BACKGROUND

In bioscience fields, activities of biological micro-objects such ascells are often studied and analyzed. For example, cells that produce atleast a minimum number of clones or secrete desired materials can beutilized in the production of medicines or in the study of diseases. Itcan thus be advantageous to identify cells that produce clones at orabove a minimum rate or that secrete certain materials. Embodiments ofthe present invention are directed to improved micro-fluidic devices andprocesses for placing selected biological micro-objects into holdingpens, conditioning the micro-objects in the pens, monitoring biologicalactivity of the micro-objects in the pens, and/or moving themicro-objects whose biological activity meets a predetermined thresholdfrom the pens for further use or processing.

SUMMARY

In some embodiments, a method of processing biological micro-objects caninclude actively placing individual biological micro-objects in interiorspaces of holding pens in a micro-fluidic device and providing a flow ofa first liquid medium to the pens over a time period. The method canalso include, while providing the flow, impeding direct flow of thefirst medium from the flow into the interior spaces of the holding pens.

In some embodiments, a micro-fluidic apparatus can include a housing andholding pens. The housing can be disposed on a base, and the housing caninclude a flow path for a first liquid medium. The holding pens can bedisposed within the housing, and each pen can comprise an enclosureenclosing an interior space. The enclosure can be structured to hold abiological micro-object suspended in a second liquid medium and impede adirect flow of the first medium into the second medium in the interiorspace.

A method of processing biological micro-objects can include creatingvirtual holding pens in a micro-fluidic device by directing a pattern oflight in the form of the holding pens into the micro-fluidic device andthereby activating dielectrophoresis (DEP) electrodes. The method canalso include placing individual biological micro-objects into theholding pens, where each of the holding pens isolates any one or more ofthe individual micro-objects in the holding pen from all of themicro-objects outside of the holding pen. The method can also includeproviding the micro-objects in the holding pens with a common flow of aliquid medium over a time period.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an example of a micro-fluidic device according tosome embodiments of the invention.

FIG. 1B is a side, cross-sectional view of the device of FIG. 1A.

FIG. 1C is a top, cross-sectional view of the device of FIG. 1A.

FIG. 2 shows a cross-sectional side view of the device of FIG. 1Aillustrating an example of the device configured with an optoelectronictweezers (OET) device according to some embodiments of the invention.

FIG. 3 is a top, cross-sectional partial view of the device of FIG. 1Aconfigured with the OET device of FIG. 2 and virtual pens according tosome embodiments of the invention.

FIG. 4 is a top, cross-sectional partial view of the device of FIG. 1Aconfigured with the OET device of FIG. 2 and pens that are physicaland/or virtual according to some embodiments of the invention.

FIG. 5A illustrates an example of a micro-fluidic structure disposed ona base that defines a fluidic channel and pens according to someembodiments of the invention.

FIG. 5B is a top, cross-sectional view of the micro-fluidic structureand base of FIG. 5A.

FIG. 6A illustrates an example of a micro-fluidic structure disposed ona base that defines fluid channels and pens according to someembodiments of the invention.

FIG. 6B is a top, cross-sectional view of the micro-fluidic structureand base of FIG. 6A.

FIG. 7 illustrates an example of a variation of the pens shown in FIG.6B according to some embodiments of the invention.

FIGS. 8A, 8B, 9, and 10 illustrate examples of alternativeconfigurations of pens according to some embodiments of the invention.

FIGS. 11A and 11B illustrated selecting and moving a micro-object usinga light trap according to some embodiments of the invention.

FIGS. 12A and 12B show selecting and moving a micro-object using avirtual barrier according to some embodiments of the invention.

FIG. 13A illustrates an example of a micro-fluidic structure disposed ona base that defines a fluid chamber and pens according to someembodiments of the invention.

FIG. 13B is a top, cross-sectional view of the micro-fluidic structureand base of FIG. 13A.

FIGS. 14 and 15 show examples of alternative configurations of the penarray of FIG. 13B according to some embodiments of the invention.

FIG. 16 shows an example of a process that includes placing biologicalmicro-objects into pens in a micro-fluidic device according to someembodiments of the invention.

FIG. 17 illustrates a process showing an example of operation of thedevice of FIG. 1A configured with the OET device of FIG. 2 according tosome embodiments of the invention.

FIGS. 18A-18C illustrate an example of processing cells in accordancewith a step of FIG. 17.

FIG. 19 illustrates an example of expanding pens as clones grown in thepens according to a step of FIG. 17.

FIG. 20 illustrates an example of turning off pens in which clones aregrowing too slowly and flushing the clones in those pens away accordingto a step of FIG. 6.

FIG. 21 illustrates an example of placing daughter clones in new pensaccording to a step of FIG. 17.

FIG. 22 illustrates a process showing another example of operation ofthe device of FIG. 1A configured with the OET device of FIG. 2 accordingto some embodiments of the invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

This specification describes exemplary embodiments and applications ofthe invention. The invention, however, is not limited to these exemplaryembodiments and applications or to the manner in which the exemplaryembodiments and applications operate or are described herein. Moreover,the Figures may show simplified or partial views, and the dimensions ofelements in the Figures may be exaggerated or otherwise not inproportion for clarity. In addition, as the terms “on,” “attached to,”or “coupled to” are used herein, one element (e.g., a material, a layer,a substrate, etc.) can be “on,” “attached to,” or “coupled to” anotherelement regardless of whether the one element is directly on, attached,or coupled to the other element or there are one or more interveningelements between the one element and the other element. Also, directions(e.g., above, below, top, bottom, side, up, down, under, over, upper,lower, horizontal, vertical, “x,” “y,” “z,” etc.), if provided, arerelative and provided solely by way of example and for ease ofillustration and discussion and not by way of limitation. In addition,where reference is made to a list of elements (e.g., elements a, b, c),such reference is intended to include any one of the listed elements byitself, any combination of less than all of the listed elements, and/ora combination of all of the listed elements.

The words “substantially” and “generally” mean sufficient to work forthe intended purpose. The term “ones” means more than one.

The term “cell” refers to a biological cell. The term “clones,” withreference to cells, means cells that are identical because each cell wasgrown from the same parent cell. Clones are thus all “daughter cells” ofthe same parent cell.

As used herein, the term “biological micro-object” includes biologicalcells and compounds such as proteins, embryos, plasmids, oocytes,sperms, genetic material (e.g., DNA), transfection vectors, hydridomas,transfected cells, and the like as well as combinations of theforegoing.

As used herein a dielectrophoresis (DEP) electrode refers to a terminalon or a region of an inner surface of a chamber for containing a liquidmedium at which DEP forces in the medium sufficient to attract or repelmicro-objects in the medium can be selectively activated anddeactivated.

The term “flow,” as used herein with reference to a liquid or gas,includes without limitation a continuous, pulsed, periodic, random,intermittent, or reciprocating flow of the liquid or gas. A “convectionflow” is a flow of a liquid or gas that is driven by pressure. A“diffusive flow” or “diffusion” is a flow of liquid or gas that isdriven by random thermal motion. The term “diffusive mixing” as usedwith respect to two or more liquid or gas media means the mixing of themedia due to spontaneous intermingling of the media as a result ofrandom thermal motion. The term “substantially,” as used herein withrespect to “convection flow,” diffusive flow,” “diffusion,” or“diffusive mixing,” means more than fifty percent.

The term “deterministic,” when used to describe selecting or placing amicro-object, means selecting or placing a specifically identified anddesired micro-object from a group of micro-objects. Deterministicallyselecting or placing a micro-object thus does not include randomlyselecting or placing merely any one of the micro-objects in a group orsub-group of micro-objects.

As used herein, the meaning of the term “processing” a micro-objectincludes any one or more of the following: moving (e.g., in a flow ofliquid medium, with an OET device, or the like), sorting, and/orselecting one or more of the micro-objects; modifying one or more of themicro-objects, wherein examples of such modifying include growingpopulations of micro-objects that are cells or other living biologicalentities, fusing two or more such micro-objects together, andtransfecting one or more micro-objects; monitoring micro-objects;monitoring growth, secretions, or the like of micro-objects that arecells or other living biological entities; and/or conditioningmicro-objects that are cells or other living biological entities.

Embodiments of the invention include deterministically placingindividual biological micro-objects in holding pens in a micro-fluidicdevice. A flow of a first liquid medium can be provided to the pens, butthe pens can be structured to impede a direct flow of the first mediuminto a second medium in the pens while allowing diffusive mixing of thefirst medium in the flow and the second medium in the pens.

FIGS. 1A-1C illustrate an example of a micro-fluidic device 100according to some embodiments of the invention. As shown, themicro-fluidic device 100 can comprise a housing 102, an electrodemechanism 108, and a monitoring mechanism 118. As also shown, thehousing 102 can comprise an interior chamber 110 for holding one or moreliquid media 114 in which a plurality of biological micro-objects 116can be suspended. The media 114 can be disposed on an inner surface 120of the chamber 110. A plurality of holding pens 112 for themicro-objects 116 can be disposed in the chamber 110. As will be seen,each pen 112 can be a virtual pen, a physical pen, and/or a combinationvirtual/physical pen.

The media 114 in the device 100 can comprise, for example, a firstmedium 122 and a second medium 124. The first medium 122 can be media114 that is in the flow path 126, and the second medium 124 can be media114 that is inside the holding pens 112. The first medium 122 can be thesame type of medium as the second medium 124. Alternatively, the firstmedium 122 can be a different type of medium than the second medium 124.

The housing 102 can comprise an enclosure that defines the chamber 110.As shown, the housing 102 can also comprise one or more inlets 104through which media 114 and micro-objects 116 can be input into thechamber 110. There can be one or more flow paths 126 in the chamber 110for the media 114. For example, as illustrated in FIG. 1C, the chamber110 can comprise a flow path 126 for media 114 from the inlet 104 to theoutlet 106.

An inlet 104 can be, for example, an input port, an opening, a valve, achannel, or the like. The housing 102 can also comprise one or moreoutlets 106 through which media 114 and micro-objects 116 can beremoved. Micro-objects 116 can alternatively be removed from the housing102 in other ways. For example, as noted below, a needle-like aspirator(not shown) can pierce the housing 102, and one or more micro-objects116 can be removed with the aspirator. An outlet 106 can be, forexample, an output port, an opening, a valve, a channel, or the like. Asanother example, the outlet 106 can comprise a droplet outputtingmechanism such as any of the outputting mechanisms disclosed in U.S.patent application Ser. No. 13/856,781 filed Apr. 4, 2013. All or partof the housing 102 can be gas permeable to allow gas (e.g., ambient air)to enter and exit the chamber 110, for example, to sustain thebiological micro-objects 116 in the chamber 110. For example, a flow ofgas can be applied to the gas permeable portion of the housing 102. Forexample, a pulsed, regulated, or otherwise controlled flow of gas can beapplied as needed (e.g., when testing indicates that micro-objects(e.g., cells) in the housing 102 need gas).

Although not shown, the device 100 can comprise sensors or similarcomponents that detect relevant conditions of media 114 or the chamber110 such as temperature, the chemical composition of media 114 (e.g.,the level of dissolved oxygen, carbon dioxide, or the like in media114), the pH of media 114, osmolarity of media 114, or the like. Thehousing 102, for example, can comprise such sensors or components, whichcan be configured with a controller (not shown) to control input ofmedia 114 through the inlet 104 to maintain constant or controllablyadjust certain conditions (such as the conditions identified above) ofmedia 114.

The electrode mechanism 108 (shown in FIG. 1B) can be configured tocreate selectively electrokinetic forces on micro-objects 116 in media114. For example, the electrode mechanism 108 can be configured toselectively activate (e.g., turn on) and deactivate (e.g., turn off)dielectrophoresis (DEP) electrodes at the inner surface 120 of thechamber 110 on which media 114 is disposed. The DEP electrodes cancreate forces in media 114 that attract or repel micro-objects 116, andthe electrode mechanism 108 can thus select and/or move one or more ofthe micro-objects 116 in media 114. For example, in some embodiments,the electrode mechanism 108 can be configured such that hardwiredelectrical connections to the DEP electrodes at the inner surface 120can activate and deactivate the individual DEP electrodes. In otherembodiments, the individual DEP electrodes at the inner surface 120 canbe optically controlled. An example comprising an optoelectronictweezers mechanism is illustrated in FIG. 2 and discussed below.

For example, the electrode mechanism 108 can include one or more optical(e.g., laser) tweezers devices and/or one or more optoelectronictweezers (OET) devices (e.g., as disclosed in U.S. Pat. No. 7,612,355,which is incorporated in its entirety by reference herein). As yetanother example, the electrode mechanism 108 can include one or moredevices (not shown) for moving a droplet of media 114 in which one ormore of the micro-objects 116 are suspended. Such devices (not shown)can include electrowetting devices such as optoelectronic wetting (OEW)devices (e.g., as disclosed in U.S. Pat. No. 6,958,132). The electrodemechanism 108 can thus be characterized as a DEP device in someembodiments.

The monitoring mechanism 118 can comprise any mechanism for observing,identifying, or detecting individual micro-objects 116 in media 114. Insome embodiments, the monitoring mechanism 118 can also comprise amechanism for monitoring biological activity or a biological state ofmicro-objects 116 in the pens 112.

As shown in FIG. 2, the monitoring mechanism 118 can comprise an imagingdevice 220. For example, the imaging device 220 can comprise a camera orsimilar device for capturing images of micro-objects 116 in the chamber110, including in the pens 112. As also shown, a controller 218 cancontrol the imaging device 220 and process images captured by theimaging device 220. Although shown as disposed below the device 102 inFIG. 2, the imaging device 220 can be disposed in other locations suchas above or to the side of the device 102.

As also shown in FIG. 2, the electrode mechanism 108 can comprise an OETdevice. For example, as shown, the electrode mechanism 108 can comprisea first electrode 204, a second electrode 210, an electrode activationsubstrate 208, and a power source 212. As shown, media 114 in thechamber 110 and the electrode activation substrate 208 can separate theelectrodes 204, 210. A pattern of light 216 from the light source 214can selectively activate a desired pattern of individual DEP electrodesat the inner surface 120 of the chamber 110. That is, light in the lightpattern 216 can reduce the electrical impedance of the electrodeactivation substrate 208 at a pattern of small “electrode” regions ofthe inner surface 120 of the chamber 110 to less than the impedance ofthe media 114. The foregoing creates an electric field gradient in themedia 114 from the electrode region of the surface 120 to the firstelectrode 204, which in turn creates local DEP forces that attract orrepel nearby micro-objects 116. Different patterns of individual DEPelectrodes that attract or repeal micro-objects 116 in media 114 canthus be selectively activated and deactivated at many different suchelectrode regions at the inner surface 120 of the chamber 110 bydifferent light patterns 216 projected form a light source 214 (e.g., alaser source or other type of light source) into the micro-fluidicdevice 100.

In some embodiments, the electrode activation substrate 208 can be aphotoconductive material, and the inner surface 120 can be featureless.In such embodiments, the DEP electrodes can be created anywhere and inany pattern on the inner surface 120 of the chamber 110 in accordancewith the light pattern 126 (see FIG. 2). Examples are illustrated in theaforementioned U.S. Pat. No. 7,612,355 in which the undoped amorphoussilicon material 24 shown in the drawings of the foregoing patent can bean example of photoconductive material that can compose the electrodeactivation substrate 208.

In other embodiments, the electrode activation substrate 208 cancomprise a circuit substrate such as a semiconductor material comprisinga plurality of doped layers, electrically insulating layers, andelectrically conductive layers that form semiconductor integratedcircuits such as known in semiconductor fields. In such embodiments,electric circuit elements can form electrical connections betweenelectrode regions at the inner surface 120 of the chamber 110 and thesecond electrode 210 that can be selectively activated and deactivatedby changing patterns of the light pattern 216. When not activated, eachelectrical connection can have high impedance such that the voltage dropfrom a corresponding electrode region at the inner surface 120 of thechamber 110 to the second electrode 210 is greater than the voltage dropfrom the first electrode 204 through media 114 to the correspondingelectrode region. When activated by light in the light pattern 216,however, each electrical connection can have low impedance such that thevoltage drop from a corresponding electrode region at the inner surface120 of the chamber 110 to the second electrode 210 is less than thevoltage drop from the first electrode 204 through media 114 to thecorresponding electrode region, which activates a DEP electrode at thecorresponding electrode region as discussed above. DEP electrodes thatattract or repeal micro-objects 116 in media 114 can thus be selectivelyactivated and deactivated at many different “electrode” regions at theinner surface 120 of the chamber 110 by the light pattern 216.Non-limiting examples of such configurations of the electrode activationsubstrate 208 include the phototransistor-based OET device 200illustrated in FIGS. 21 and 22 of U.S. Pat. No. 7,956,339.

In some embodiments, the first electrode 204 can be part of an upperwall 202 of the housing 102, and the electrode activation substrate 208and second electrode 210 can be part of a lower wall 206 of the housing102 generally as illustrated in FIG. 2. As shown, the upper wall 202 andlower wall 206 can define the chamber 110, and media 114 can be disposedon the inner surface 120 of the chamber 110. The foregoing, however, isbut an example. In other embodiments, the first electrode 204 can bepart of the lower wall 206 and one or both of the electrode activationsubstrate 208 and/or second electrode 210 can be part of the upper wall202. As another example, the first electrode 204 can be part of the samewall 202 or 206 as the electrode activation substrate 208 and the secondelectrode 210. For example, the electrode activation substrate 208 cancomprise the first electrode 204 and/or the second electrode 210.Moreover, the light source 214 can alternatively be located below thehousing 102, and/or the imaging device 220 and the light source 214 canalternatively be located on the same side of the housing 102.

As mentioned, in some embodiments of the invention, part or all of eachpen 112 can be “virtual,” which as used herein, means that part or allof the pen 112 comprises DEP forces from activated DEP electrodes atelectrode regions of the interior surface 120 of the chamber 110 (asdiscussed above) rather than physical barriers.

FIG. 3 (which shows a partial, top cross-sectional view of part of thehousing 102) illustrates an example of the device 100 of FIGS. 1A-1C inwhich the pens 112 (which are designated 302 in FIG. 3) are virtual pens302 according to some embodiments of the invention. The virtual pens 302in FIG. 3 can be created in the chamber 110 by the electrode mechanism108 configured, for example, as the OET device of FIG. 2. That is, thevirtual pens 302 can comprise a pattern of activated DEP electrodes atthe inner surface 120 of the chamber 110. Although one micro-object 116is shown in each pen 302, there can alternatively be more than onemicro-object 116 in each pen.

As shown in FIG. 3, a flow 314 of media 114 through the chamber 110 canbe provided in a flow path 126. As illustrated in FIG. 3, each pen 302can isolate the micro-object(s) 116 in the pen 302 from themicro-objects 116 in the other pens 302. The flow 314 of media 114,however, can be a common flow 314 provided to some or all of the pens302 and thus the micro-objects 116 in the pens 302. Configured as shownin FIG. 3, each pen 302 can thus isolate the micro-object(s) 116 insidethe pen 302 from micro-objects 116 outside of the pen 302 includingmicro-objects 116 in other pens 302 and thus prevent a micro-object 116from outside of a particular pen 302 from mixing with themicro-object(s) inside that particular pen 302 while allowing a commonflow 314 of media 114 to flow into (by convection flow) and out ofmultiple pens 116 and thus, for example, supply nutrients and carry awaywaste from micro-objects 116 in multiple pens 116.

The virtual pens 302 can comprise light enclosures in the light pattern216 projected by the light source 214 into the housing 102 of themicro-fluidic device 100 as shown in FIG. 2. The power source 212 of theOET of FIG. 2 can be configured with a frequency that causes the lightenclosure that defines each pen 302 to repel a micro-object 116 so thateach pen 302 holds a micro-object 116 inside the pen 302. Moreover, oneor more of the virtual pens 302 can be moved, expanded or contracted,turned off, or the like by changing the light pattern 216 projected intothe housing 102.

As shown in FIG. 3, the OET device depicted in FIG. 2 can also create alight trap 304 (e.g., cage) that traps a micro-object 116 to select andmove the micro-object 116. The light trap 304 can be, for example, alight cage that traps the micro-object 116. The frequency of the powersource 212 in FIG. 2 can be such that the light trap 304 repels theselected micro-object 116. The micro-object 116 can thus be moved in thechamber 110 by moving the light trap 304 on the electrode activationsubstrate 208. The detector 220 can capture images of the micro-objects116 in the channel 110 (e.g., a flow path 126), which can be an exampleof a common space. Specific, desired individual ones of themicro-objects 116 can thus be identified and then selected with theselector 118 (e.g., configured as the OET device of FIG. 2), forexample, with light traps 302, 412 as discussed below with respect toFIGS. 3 and 4. The detector 220 and selector 118 (e.g., configured asthe OET device of FIG. 2) can thus be examples of a means fordeterministically selecting or placing one or more of the micro-objects116.

Although illustrated as squares in FIG. 3, the pens 302 canalternatively be other shapes. For example, the pens 302 can be circles,ovals, rectangles, triangles, or the like. Moreover, the pens 302 neednot be fully enclosed. For example, any of the pens 302 can have anopening 308 as illustrated by pen 302 a in FIG. 3. Although illustratedas a circle, the light trap 304 can be other shapes such as square,oval, rectangular, triangular, or the like. In addition, the pens 302can be difference sizes and can be disposed in different orientations.

FIG. 4 (which shows a partial, top cross-sectional view of part of thehousing 102) illustrates another example of a configuration of thedevice 100 of FIGS. 1A-1C. In the configuration illustrated in FIG. 4,the pens 112 (which are designated 402 in FIG. 4) can be entirelyphysical or both physical and virtual. For example, as shown, each pen402 can comprise a physical barrier 404 (e.g., as part of the housing102), which can define or be part of an enclosure 406 with an opening408 that is in fluidic communication (e.g., contact) with a flow 314 ofmedia 114 through the chamber 110.

Generally as discussed with respect to FIG. 3, a flow 314 of media 114through the chamber 110 can be provided in a flow path 126. Each pen 402can isolate the micro-object(s) 116 in the pen 402 from themicro-objects 116 in the other pens 302. For example, each pen 302 canprevent any micro-object 116 outside the pen 302 from mixing with any ofthe micro-objects inside the pen 302. The flow 314 of media 114,however, can be a common flow 314 provided to all of the pens 402 andthus all of the micro-objects 116 in the pens 402. The pens 402,however, can be structured so that the first medium 122 from the flow314 does not flow directly into any of the pens 402, but the structureof the pens 402 can allow diffusive mixing of the first medium 122 fromthe flow 314 and the second medium 124 inside the pens 402.

For example, the barrier 404 of each pen 402 can be shaped and orientedto impede direct flow of the first medium 122 from the flow 314 in theflow path 126 into the pen 402. For example, each pen 402 can be shapedand oriented such that a portion of the physical barrier 404 directlyfaces the direction of the flow 314 but no opening (e.g., the opening408) directly faces the direction of the flow 314. In the exampleillustrated in FIG. 3, each of the pens 402 thus impede direct flow ofthe first medium 122 from the flow 314 in the flow path 126 into the pen402.

As another example, the barrier 404 can be shaped and oriented toprevent convection flow of the first medium 122 from the flow 314 in theflow path 126 into the pen 402. Each pen 402 can, however, be shaped andoriented to allow substantially only diffusion mixing of the firstmedium 122 from the flow 314 in the flow path 126 and the second medium424 inside the pen 402. For example, each pen 402 can comprise anopening shaped and oriented to allow such diffusive mixing.

In some embodiments, however, the pens 402 can be oriented with theopening 408 pointed in any direction with respect to the flow 314 ofmedia 114. As also shown, any of the pens 402 can comprise both aphysical barrier and a virtual portion. For example, in someembodiments, a virtual door 410 comprising adjacent activated DEPelectrodes on the inner surface 120 of the chamber 110 can be createdand/or removed at the opening 408 of one or more of the physicalbarriers 404 to make the pen 402 selectively fully enclosed generally asshown in FIG. 4. The virtual door 410 can correspond to light in thelight pattern 214 projected onto the electrode activation substrate 208.(See FIG. 2.)

As also illustrated in FIG. 4, one or more of the pens 402 can comprisemore than one such virtual door 410. For example, as shown, pen 402 acomprises more than one opening 408 a, 408 b into the pen 402 a, andthere can be a virtual door 410 a, 410 b at each such opening 408 a, 408b. In operation, a micro-object 116 can be moved into the pen 402 athrough the first opening 408 a while the first virtual door 410 a isturned off, and the micro-object 116 can later be moved out of the pen402 a through the second opening 408 b while the second virtual door 410b is turned off.

A light trap 412 (which can be similar to or the same as light trap 304)can be created on the surface 120 of the chamber 110 by the electrodemechanism 108 configured as the OET device of FIG. 2. The light trap 412can be created that traps a micro-object 116 to select the micro-object116. The frequency of the power source 212 in FIG. 2 can be such thatthe light trap 412 repels the selected micro-object 116. Themicro-object 116 can thus be moved in the chamber 110 by moving thelight trap 412 on the photoconductive layer 308. For example, amicro-object 116 can be selected and moved into and/or output of a pen402 by forming a light trap 412 that traps the micro-object 116 and thenmoving the light trap 412 on the inner surface 102.

Although illustrated as partial squares in FIG. 4, the pens 402 canalternatively be other shapes. For example, the pens 402 can be partialcircles, ovals, rectangles, triangles, or the like. The light trap 412can similarly have other shapes than the circle shown.

Like pens 302 and 402, the pens 112 can, in some embodiments, also beconfigured to impede direct flow (e.g., convection flow) of the firstmedium 122 from a common flow in a flow path 126 into the pens 112 whileallowing substantially only diffusive mixing of the first medium 122from the common flow 314 in a flow path 126 and the second medium insidea pen 112.

The housing 102, however, need not be configured with a single commonspace for media 114. Rather, the housing 102 can comprise one or moreinterconnected chambers, channels, or the like for containing media 114and through which media 114 can flow. FIGS. 5A-7 illustrate examples.

As shown in FIGS. 5A and 5B, the housing 102 of the device 100 (seeFIGS. 1A-1C) can comprise a base (e.g., a substrate) 502 on which isdisposed one or more micro-fluidic structures 500. The base 502 cancomprise, for example, the lower wall 206 as discussed above withrespect to FIG. 2, and all or part of the top surfaces of themicro-fluidic structure 500 can comprise the upper wall 202 includingany variation discussed above.

As shown, the micro-fluidic structure 500 can comprise a channel 504 andpens 506, each of which can comprise an enclosure 510 and an opening 508to the channel 504. As shown, the pens 506 and the channel 504 can bethe same or a different height from the base 502. The channel 504 andpens 506 can correspond to the chamber 110 of FIGS. 1A-1C and 2, and thesurface 522 of the base 502 can correspond to the inner surface 120 ofthe chamber 110 of FIGS. 1A-1C and 2. Thus, in embodiments of theinvention in which the housing 102 comprises the base 502 and the OETdevice of FIG. 2, DEP electrodes can be activated and deactivated inaccordance with the light pattern 216 at the surface 522 of the base 502rather than the inner surface 120 of the chamber 110.

One or more micro-objects 116 can be deterministically selected andmoved (e.g., using the detector 220 and selector 118 as discussed above)from the channel 504 (which can be an example of a common space and/or aflow path) through the opening 508 into the enclosure 510 of a pen 506.The micro-object(s) 116 can then be held for a period of time in the pen506. The opening 508 and enclosure 510 of each pen can be sized andconfigured and the rate of the flow 520 of media 114 in the channel 504can be such that the flow 520 creates little to no appreciableconvection inside the enclosure 510. Once placed in a pen 506,micro-object(s) 116 thus tend to stay in the pen 506 until activelyremoved from the pen 506. Diffusion through the opening 508 betweenmedia 114 in the channel 504 and the enclosure 510 can provide forinflow into the enclosure 510 from the channel 504 of nutrients for themicro-object(s) 116 in a pen 506 and outflow from the enclosure 510 intothe channel 504 of waste from the micro-object(s) 116.

The pens 506 can be structured so that a first medium 122 in the flow520 in the channel 504 does not flow directly into any of the pens 506,but the structure of the pens 506 allows diffusive mixing of the firstmedium 122 from the flow 520 through the opening 508 in the pen 506 witha second medium 124 inside the pen 506 generally as discussed above.

The channel 504 and the pens 506 can be physical structures as shown inFIGS. 5A and 5B. For example, the micro-fluidic structure 500 cancomprise a flexible material (e.g. rubber, plastic, an elastomer,polydimethylsioxane (“PDMS”), or the like), which can also be gaspermeable in some embodiments. Alternatively, the micro-fluidicstructure 500 can comprise other materials including rigid materials.Although one channel 504 and three pens 506 are shown, the micro-fluidicstructure 500 can comprise more than one channel 504 and more or fewerthan three pens 506. As shown in FIG. 5B, a virtual door 512 canoptionally be created and removed closing and opening the opening 508 ofeach of the pens 506. Such virtual doors 512 can be created byactivating DEP electrodes at the surface 522 of the base 502 generallyas discussed above with regard to the inner surface 120.

Although the channel 504 and pens 506 are illustrated in FIGS. 5A and 5Bas physical, the channel 504 and pens 506 can alternatively be virtual.For example, all or part of the channel 504 and/or the pens 506 can becreated by activating DEP electrodes at the surface 522 of the base 502generally as discussed above.

In the example shown in FIGS. 6A and 6B, the housing 102 of the device100 (see FIGS. 1A-1C) can comprise the base 502 of FIGS. 5A and 5B and amicro-fluidic structure 602 disposed on the surface 522 of the base 502.As can be seen in FIG. 6B, the micro-fluidic structure 602 can comprisea pen structure 612, which can comprise pens 606. Each such pen 606 cancomprise an enclosure 610 in which a micro-object 116 can be placed andheld for a time period. As also shown in FIG. 6B, the micro-fluidicstructure 602 can define channels 604, and the opening 608 of each pen606 can be in fluidic communication (e.g., contact) with media 114 inone of the channels 604.

One or more micro-objects 116 can be deterministically selected (asdiscussed above) and moved from one of the channels 604 (which can be anexample of a common space and/or a flow path) through the opening 608into the enclosure 610 of a pen 606. The micro-object(s) 116 can then beheld in a pen 606 for a period of time. Thereafter, the micro-object(s)116 can be moved from the enclosure 610 through the opening 608 into thechannel 604. Flows 620 of media 114 in the channels 604 can movemicro-objects 116 in the channels 604.

Because the openings 608 of the pens 606 are in fluidic communicationwith a channel 604, the flows 620 of media 114 in the channels 604 canprovide nutrients to the micro-objects 116 in the pens 606 and allow forthe outflow of waste from the micro-objects 116 during the period oftime that the micro-objects 116 are held in the pens 606. The flows 620in the channels 604 can thus constitute a common flow of media 114 tothe pens 606, which like pens 506, can otherwise physically separate andisolate micro-objects 116.

The pens 606 can be structured so that a first medium 122 in a flow 620in a channel 604 does not flow directly into any of the pens 606, butthe structure of the pens 606 allows diffusive mixing of the firstmedium 122 from a flow 620 through an opening 608 in the pen 606 with asecond medium 124 in a pen 606. For example, a pen 606 can be physical(rather than virtual) and the opening 608 of the pen 606 can be orientedin any direction so long as no part of the opening 608 faces directlyinto a flow 620. A pen 606 can thus impede direct flow of the firstmedium 122 into the pen 606.

The pens 606 can be physical structures as shown in FIG. 6B. Forexample, the micro-fluidic structure 600 can comprise any of thematerials discussed above with respect to the micro-fluidic structure500 of FIGS. 5A and 5B. Although two channels 604 and twelve pens 606are shown in FIG. 6B, the micro-fluidic structure 602 can comprise moreor less than two channels 604 and more or fewer than twelve pens 606.Although not shown, a virtual door like door 512 of FIG. 5B canoptionally be created at the openings 608 of one or more of the pens606.

Although the micro-fluidic structure 602 including the pen structure 612are shown in FIGS. 6A and 6B as physical, all of part of the structure602 can alternatively be virtual and thus created by activating DEPelectrodes at the surface 522 of the base 502 as discussed above withrespect to the inner surface 120. For example, all or part of the penstructure 612 can be virtual rather than physical.

FIG. 7 is similar to FIG. 6B except that a channel 704 (which can be anexample of a flow path 126) is disposed between pen structures 712 asshown. Otherwise, each pen 706 can be similar to each pen 606. Forexample, each pen 706 can comprise an enclosure 710 in which amicro-object 116 can be placed and held. As also shown in FIG. 7, theopening 708 of each pen 706 can be in fluidic communication (e.g.,contact) with media 114 in the channel 704. One or more micro-objects116 can be deterministically selected (as discussed above) and movedfrom the channel 704 (which can be an example of a common space) throughthe opening 708 into the enclosure 710 of a pen 706, where themicro-object(s) 116 can be held for a period of time. Thereafter, themicro-object(s) 116 can be moved from the enclosure 710 through theopening 708 into the channel 704. The flow 720 of media 114 in thechannels 704 can move micro-objects 116 in the channel 704.Alternatively or in addition, the micro-objects 116 can be moved by DEPforces, centrifugal forces, and/or the like.

Because the openings 708 of the pens 706 are in fluidic communicationwith the channel 704, the flow 720 of media 114 in the channel can alsoprovide nutrients to the micro-objects 116 in the pens 706 and providefor the outflow of waste from the micro-objects 116 during the period oftime that the micro-objects 116 are held in the pens 706. The flow 720in the channel 704 can thus constitute a common flow of media 114 to allof the pens 706.

The pens 706 can be structured so that a first medium 122 in the flow720 in the channel 704 does not flow directly into any of the pens 706,but the structure of the pens 706 allows diffusive mixing of the firstmedium 122 in the channel 704 through an opening 708 in the pen 706 withsecond medium 124 in a pen 706. For example, a pen 706 can be physicaland can be oriented so that no opening to the pen 706 faces directlyinto the flow 720.

Although one channel 704 and twelve pens 706 are shown in FIG. 7, therecan be more or fewer. Although not shown, a virtual door like door 512of FIG. 5B can optionally be created at the openings 708 of one or moreof the pens 706. Although the pen structures 712 are shown in FIG. 7 asphysical, all or part of the pen structures 702 can alternatively bevirtual and thus created by activating DEP electrodes at the surface 522of the base 502 as discussed above with regard to inner surface 120.

The shape and configuration of the pens 506, 606, 706 (or any pendisclosed herein) illustrated in FIGS. 5A-7 are examples only, and thosepens 506, 606, 706 (or any pen disclosed herein) can take other shapesand/or configurations. For example, any of pens 506, 606, 706 (or anypen disclosed herein) can be circular, oval, triangular, or the likerather than square or rectangular. As other examples, any of the pens506, 606, 706 (or any pen disclosed herein) can be replaced by the pen806, 826, 906, 926 illustrated in FIGS. 8A-10.

As shown in FIG. 8A, a pen 806 can comprise an opening 812 (e.g.,corresponding to openings 506, 606, 706) that is smaller than the fullwidth of the enclosure 810 (e.g., corresponding to enclosures 510, 610,710). As also shown in FIG. 8A, a pen 806 can comprise one or moresecondary openings 814 (one is shown but there can be more). The opening812 can be larger than a micro-object 116 (not shown in FIG. 8A), andthe secondary opening 814 can be smaller than a micro-object 116. Thesecondary opening 814 can allow, for example, media 114 (not shown inFIG. 8A) to flow into or out of the pen 806. For example, media 114 canflow into the pen 806 through the opening 812 and out of the pen 806through the secondary opening 814. As also shown in FIG. 8A, the wallsof a pen need not be the same thickness.

As shown in FIG. 8B, a pen 826 can comprise an inner wall 834 thatextends from an opening 832 (e.g., corresponding to openings 508, 608,708, 812) to create an inner containment space 836 within the enclosure840 (e.g., corresponding to enclosures 510, 610, 710).

As illustrated in FIG. 9, a pen 906 can comprise one or more additionalpens 916 (one is shown but there can be more). For example, one or moreinner pens 916 (one is shown but there can be more) comprising anopening 922 and an enclosure 920 can be disposed inside the enclosure910 of an outer pen 906, which can comprise opening 912. One or moremicro-objects 116 (not shown in FIG. 9) can be disposed in the enclosureof each inner pen 916 and the outer pen 906.

As shown in FIG. 10, a pen 926 (comprising an opening 932 and enclosure930) can comprise multiple holding spaces 936 (although three are shown,there can be more or fewer) separated by interior walls 934. One or moremicro-objects 116 (not shown in FIG. 10) can be disposed in each holdingspace 936. For example, a different type of micro-object 116 can bedisposed in each holding space 936.

Any of the pens disclosed herein can be configured to be like or to haveany of the characteristics of the pens 806, 826, 906, 926 illustrated inFIGS. 8A-10.

Regardless of the configuration of the pens, micro-objects 116 can bedeterministically selected and moved from the flows 520, 620, 720 in thechannels 504, 604, 704 into pens 506, 606, 706 in FIGS. 5A-7 (includingthe variations of the pens 506, 606, 706 illustrated in FIGS. 8A-10) byany of a variety of mechanisms. FIGS. 11A-12B illustrate examples inwhich the OET device of FIG. 2 is used to do so. In FIGS. 11A-12B, thechannel 1104 can be any of the channels 504, 604, 704; the pen 1106 canbe any of the pens 506, 606, 706; and the flow 1120 of media 114 can beany of the flows 520, 620, 720 in FIGS. 5A-7.

As shown in FIG. 11A, a micro-object 116 can be deterministicallyselected in the flow 1120 in the channel 1104 by creating a light trap1108 (e.g., like light trap 304) that traps the micro-object 116, whichcan trap the micro-object 116 in the trap 1108. As shown in FIG. 11B,the light trap 1108 can then be moved from the channel 1104 into the pen1106, where the micro-object 116 can be released from the light trap1108. The light trap 1108 can be like and can be created and moved onthe surface 522 of the base 502 by the OET device of FIG. 2 in the sameway as light traps 304, 412 are created and moved on the inner surface120 as discussed above.

As shown in FIG. 12A, a micro-object 116 can be deterministicallyselected in the flow 1120 in the channel 1104 by creating a virtualbarrier 1208 in the path of the micro-object 116 in the channel 1104. Asillustrated in FIG. 12B, the virtual barrier 1208 can deflect themicro-object 116 into the pen 1106. The virtual barrier 1208 can becreated by activating DEP electrodes on the surface 522 of the base 502using the OET device of FIG. 2 generally as discussed above. Once theselected micro-object 116 is deflected into the pen 1106, the virtualbarrier 1208 can be removed from the channel 1104.

As mentioned above, micro-objects 116 can be contained in any of thepens disclosed herein for a period of time after which the micro-objects116 can be removed from the pens. In some embodiments, micro-objects 116can be removed from pens in any of the ways illustrated in FIGS.11A-12B.

For example, a light trap 1108 can be formed that traps a micro-object116 in a pen 1106 and the light trap 1108 can be moved out of the pen1106 into the channel 1104, which is the reverse of the process shown inFIGS. 11A and 11B. Once in the channel 1104, the light trap 1108 can beturned off, releasing the micro-object 116 into the flow 1120 of media114 in the channel 1104.

As another example, a virtual barrier similar to the barrier 1208 shownin FIGS. 12A and 12B can be formed in a pen 1106 to nudge a micro-object116 out of the pen 1106 into the flow 1120 of media 114 in the channel1104. The foregoing is the reverse of the process shown in FIGS. 12A and12B.

As yet another example, any of the physical pens disclosed herein can beconfigured like the outputting mechanisms 800 disclosed in theaforementioned U.S. patent application Ser. No. 13/856,781. In such aconfiguration, the pens can be configured like the expressing mechanism804 in the foregoing patent application, and a striking mechanism (notshown) like the striking mechanism 802 in the foregoing patent can beprovided to express the micro-objects 116 from the pens.

FIGS. 13A and 13B illustrate a micro-fluidic device 1300 that can be anexample of the device 100 of FIGS. 1A-1C in which the base 502 and amicro-fluidic structure 1302 are examples of the housing 102, thechamber 1308 is an example of the chamber 110, the inlet 1314 is anexample of the inlet 104, the outlet 1316 is an example of the outlet106, and the pens 1306 are examples of the pens 112. (Compare to FIGS.1A-1C.)

As shown in FIGS. 13A and 13B, the device 1300 can comprise amicro-fluidic structure 1302 disposed on the base 502 (which isdescribed above with respect to FIGS. 5A and 5B). As can be seen in FIG.13B, the micro-fluidic structure 1302 and base 502 can define a chamber1308 for media 114 and micro-objects 116. Media 114 with micro-objects116 can be input into the chamber 1308 through an inlet 1314 and outputfrom the chamber 1308 through an outlet 1316. A flow 1320 of media 114can thus be provided in the chamber 1308 from the inlet 1314 to theoutlet 1316. The inlet 1314 and outlet 1316 can be the same as orsimilar to the inlet 104 and outlet 106 of FIGS. 1A-1C as discussedabove. The channels 1304 are examples of common spaces and/or flow pathsfor media 114.

As also shown in FIG. 13B, a gas exchanger 1310 and an array 1312 ofpens 1306 and channels 1304 can be disposed in the chamber 1308 betweenthe inlet 1314 and the outlet 1316 and thus in the flow 1320 of media114. The flow 1320 of media 114 can thus pass from the inlet 1314through the gas exchanger 1310, through the channels 1304 of the penarray 1312, and out the outlet 1316. Alternatively, the inlet 1314 canbe located between the gas exchanger 1310 and the pens 1304, and the gasexchanger 1310 can thus be located upstream from the inlet 1314.

The channels 1304 and pens 1306 can be like any of the channels and pensdiscussed herein. For example, the channels 1304 can be like any ofchannels 504, 604, 704, 1104, 1204 including any variation of thosechannels discussed above, and the pens 1306 can be like any of pens 112,302, 402, 506, 606, 706, 806, 906, 1106, 1206 including any variation ofthose pens discussed above.

Openings of the pens 1306 can be in fluidic communication (e.g.,contact) with one of the channels 1304. As micro-objects 116 (not shownin FIGS. 13A and 13B) move with the flow 1320 of media 114, ones of themicro-objects 116 can be selected in a channel 1304 and moved into a pen1306. A micro-object 116 can be deterministically selected in a channel1304 and moved into a pen 1306 using any technique or mechanismdiscussed above (e.g., with light traps like light traps 304, 412, 1108;with a virtual barrier like barrier 1208; or the like). The flow 1320 ofmedia 114 can also be a common flow that carries nutrients to andprovides for the outflow of waste from micro-objects 116 in the pens1306, which can otherwise isolate micro-objects 116 from each other.Moreover, each of the pens 1306 can be structured so that media 114(e.g., the first medium 122 shown in FIGS. 1B and 1C) in a flow 1320 ina channel 1304 does not flow directly into any of the pens 1306, but thestructure of each pen 130 can allow diffusive mixing of media 114 from aflow 1320 in a channel 1304 and media 114 (e.g., the second medium 124shown in FIGS. 1B and 1C) in a pen 1306 generally as discussed above.

The configuration of the pen array 1312 in FIG. 13B is but an example.FIGS. 14 and 15 illustrate examples of alternative configurations.

As shown in FIG. 14, a pen array 1400 can comprise rows of pens 1402,and openings of the pens 1402 can be in fluidic communication (e.g.,contact) with a single channel 1404. The pen array 1400 and channel 1404can replace the pen array 1312 and channels 1304 in FIG. 13B, and theflow 1320 of media 114 in FIG. 13B can be through the channel 1404.

The pen array 1500 and channels 1504 in FIG. 15 can also replace the penarray 1312 and channels 1304 in FIG. 13B. As shown in FIG. 15, the penarray 1500 can comprise rows of pens 1502 with openings in directfluidic communication with channels 1504 c. A plurality of firstbranching channels 1504 b can connect an input channel 1504 a to thechannels 1504 c that flow directly past the pens 1502. Other (second)branching channels 1504 d can connect the channels 1504 c to an outputchannel 1504 e. The flow 1320 of media 114 in FIG. 13B can be into thefirst channel 1504 a, through branching channels 1504 b to the channels1504 c in direct fluidic communication with the pens 1502, through otherbranching channels 1504 d to the second channel 1504 e.

The channels 1404, 1504 in FIGS. 14 and 15 can be like channels 1304 asdiscussed above. The pens 1402, 1502 can likewise be like pens 1306 asdiscussed above. The channels 1404, 1504 can be examples of commonspaces and/or flow paths. Each pen 1402, 1502 can be structured so thatmedia 114 (e.g., the first medium 122 in FIGS. 1B and 1C) in a flow in achannel 1404, 1504 does not flow directly into the pen 1402, 1502, butthe structure of each pen 1402, 1502 can allow diffusive mixing of mediafrom a flow in a channel 1404, 1504 and media (e.g., the second medium124 in FIGS. 1B and 1C) in a pen 1402, 1502 generally as discussedabove.

FIG. 16 illustrates an example of a process 1600 for processingbiological micro-objects in pens. The process 1600 can be performedusing any of the micro-fluidic devices discussed above or similardevices. For example, the process 1600 can be performed using themicro-fluidic devices 100 and 1300 including any variation of thosedevices discussed above (e.g., as illustrated in FIGS. 2-12B, 14, and15).

As shown in FIG. 16, at step 1602, the process 1600 can load biologicalmicro-objects into a micro-fluidic device. For example, the process 1600can introduce into the chamber 110 of the device 100 of FIGS. 1A-1Cthrough the inlet 104 micro-objects 116 in media 114. As anotherexample, the process 1600 can introduce into the chamber 1308 of thedevice 1300 of FIGS. 13A and 13B micro-objects 116 in media 114 throughthe inlet 1314.

At step 1604, the process can select individual ones of the biologicalmicro-objects loaded at step 1602. For example, the process 1600 canselect a sub-set of less than all of the micro-objects 116 in media 114that have a particular characteristic. The micro-objects 116 can bemonitored, for example, using the imaging device 220 of FIG. 2. At step1604, one micro-object 116 having a particular desired characteristiccan be deterministically selected and loaded into one pen such that step1604 results in one and only one micro-object 116 in each of a pluralityof the pens. Alternatively, more than one micro-object 116 can be loadedinto a pen.

At step 1606, the process 1600 can place the micro-objects 116 selectedat step 1604 into pens of the micro-fluidic device. For example, at step1606, the process 1600 can place selected micro-objects 116 into thepens 112, 302, 402, 506, 606, 706, 806, 906, 1106, 1206, 1306, 1402,1502 using any of the techniques discussed above. As noted above andillustrated throughout the drawings, the foregoing pens can physicallyseparate micro-objects 116 one from another. That is, each pen canphysically separate the micro-object 116 or micro-objects 116 in the penfrom all other micro-objects 116 in the micro-fluidic device 100, 1300.After placing selected micro-objects 116 in the pens at step 1606, theprocess 1600 can keep the micro-objects 116 in the pens for a timeperiod.

At step 1608, the process 1600 can provide a flow of liquid media 114 tothe pens. Step 1608 can be accomplished by providing any of the flows314, 314, 520, 620, 720, 1120, 1320 in the chambers 110, 1308 orchannels 504, 604, 704, 1104 as discussed. It is noted that, at step1606, individual micro-objects 116 can be physically isolated from eachother by being placed in physically separated pens, but at step 1608,those micro-objects 116 in the pens can be provided with the same flowof media 114. As noted above, the pens 112, 302, 402, 506, 606, 706,806, 906, 1106, 1206, 1306, 1402, 1502 can be structured to impededirect flow of media 114 (e.g., the first medium 122 shown in FIGS. 1Band 1C) from the flows 314, 314, 520, 620, 720, 1120, 1320 in thechambers 110, 1308 or channels 504, 604, 704, 1104 into the pens 112,302, 402, 506, 606, 706, 806, 906, 1106, 1206, 1306, 1402, 1502 whileallowing diffusive mixing of media 114 (e.g., the first medium 122 shownin FIGS. 1B and 1C) from the flows 314, 314, 520, 620, 720, 1120, 1320and media 114 (e.g., the second medium 124 shown in FIGS. 1B and 1C)inside the pens.

As mentioned, the micro-objects 116 placed into the pens at step 1606can be kept in the pens for a time period during which step 1608 canprovide the micro-objects 116 with the flow of media 114, which throughthe diffusive mixing discussed above can provide the micro-objects 116in the pens with nutrients and provide for the outflow of waste from themicro-objects 116. At step 1610, the process 1600 can monitor one ormore biological activities of the micro-objects 116 in the pens.Examples of such biological activities can include clone production,secretion of certain biological substances, or the like. The monitoringat step 1610 can be continuous during the time period, periodicallyduring the time period, at the end of the time period, or the like. Themonitoring at step 1610 can be performed in any manner suitable foranalyzing biological activities of the micro-objects 116. For example,the monitoring at step 1610 can be performed using the imaging system220 of FIG. 2, with sensors (not shown) in or adjacent the pens, or thelike.

At step 1612, the process 1600 can select the micro-objects 116 in thepens that meet a predetermined criteria, threshold, or conditionassociated with the biological activity or state monitored at step 1610.The micro-objects 116 selected at step 1612 can be removed from the pensfor further processing or use. For example, the selected micro-objects116 can be removed from the pens using any technique or processdiscussed above. As another example, one or more micro-objects 116 canbe removed from a pen by piercing the housing with a needle-likeaspirator (not shown), and removing the micro-objects 116 with theaspirator. A specific, controlled number of micro-objects 116 can beremoved, for example, by selecting and removing that number ofmicro-objects 116 or, if the micro-objects 116 are biological cells,removing all of the cells when a colony of cloned cells reaches thedesired number.

At step 1614, the process 1600 can discard the micro-objects 116 notselected at step 1612, which are the micro-objects 116 that do not meetthe predetermined criteria, threshold, or condition associated with thebiological activity or state monitored at step 1610.

FIG. 17 illustrates an example process 1700 for growing colonies ofcloned cells from a single parent cell according to some embodiments ofthe invention. The process 1700 can be an example of the process 1600 ofFIG. 16. For example, the process 1700 can start after steps 1602 and1604 of FIG. 16 are performed; steps 1702-1706 can be performed duringsteps 1606 and 1608; step 1708 can be an example of step 1610; step 1710can be an example of step 1614; and step 1712 can be an example of step1612.

For ease of illustration and discussion the process 1700 is discussedbelow as performed with the device 100 configured with the OET device ofFIG. 2 for creating and manipulating the virtual pens 302 of FIG. 3. Theprocess 1700, however, can be performed with other configurations of thedevice 100 or the device 1300 in which the pens are virtual pens.

As shown in FIG. 17, at step 1702, the process 1700 can process cells inpens. Such processing can include fusing two cells into one cell,transfecting a cell by injecting a biological vector into a cell, or thelike. FIGS. 18A-18C illustrate an example.

As shown in FIG. 18A, two different types of micro-objects 116 and 1804can be placed in media 114 in the chamber 110. The OET device of FIG. 2can generate light traps 1806, 1808 (e.g., like light trap 304) toselect one of the first cell type 116 and one of the second cell type1804. The light traps 1806 and 1808 can then be moved into contact suchthat the first cell type 116 and the second cell type are in contact asshown in FIG. 18B. Such paired cells 1810 can then be subjected to oneor more treatments (e.g., including in the flow 314 a fusing chemical(e.g., polyethylene glycol (PEG), the Sendai virus, piercing themembranes of one of the cells 116, 1804, electric fields, pressure, orthe like)) that fuse the paired cells 1810 together to form a fused cell1812 as shown in FIG. 18C. That is, each fused cell 1812 can compriseone of the first cell types 116 and one of the second cell types 1804fused together. The light traps 1806 and 1808 can be like and can becreated and manipulated like the light trap 304, 412 as discussed aboveincluding any variation thereof.

As also shown in FIG. 18C, individual fused cells 1812 can be placed invirtual pens 1814, 1816, 1818, 1820. Although four pens 1814, 1816,1818, 1820 are shown, there can be more or fewer. The virtual pens 1814,1816, 1818, 1820 can be the same as or similar to the pens 302 of FIG. 3as described above.

Alternatively, element 1804 in FIG. 18A can be a biological vector to betransfected into a micro-object 116. In such a case, each cell 1812 inFIG. 18C can be one of the micro-objects 116 transfected with a vector1804.

As yet another alternative, the cells 1812 in FIG. 18C (whether fusedcells or transfected cells) can be processed in another device and thenplaced into the pens 1814, 1816, 1818, 1820. In such an alternative,step 1702 is not included in the process of FIG. 17. As a still furtheralternative, cells 1812 can be simple cells rather than fused ortransfected cells.

Referring again to FIG. 17, at step 1704, clones can be grown in eachpen 1814, 1816, 1818, 1820 from the cell 1812 in the pen. This can befacilitated by including a growth medium in the flow 314 through thechamber 114. At step 1706, the pens 1814, 1816, 1818, 1820 can beexpanded as the clones grow in each pen. FIG. 19 illustrates an example.As shown in FIG. 19, as the number of cells 1812 in each pen 1814, 1816,1818, 1820 increases, the size of the pens 1814′, 1816′, 1818′, 1820′can be expanded to accommodate the growing clone populations in eachpen.

At step 1708 of FIG. 17, each pen 1814, 1816, 1818, 1820 can be examinedand clone growth in the pen can be analyzed. For example, a fluorescentlabel (e.g., a biological fluorescent compound that fluoresces whenstimulated or otherwise) that binds to the clones can be included in theflow 314 through the chamber 110. The level that each pen 1814, 1816,1810, 1820 fluoresces can then be analyzed to determine clone growth ineach pen.

At step 1710, the clones in the pens 1814, 1816, 1818, 1820 in which theclones 1812 are growing at less than a minimum amount (or are otherwiseundesirable) can be discarded. FIG. 20 illustrates an example. Forpurposes of the example illustrated in FIG. 20, it is assumed that atstep 1710 it was determined that the clones 1812 in pens 1814′, 1820′ ofFIG. 19 grew less than a minimum threshold amount and are to bediscarded. As shown in FIG. 20, the pens 1814′, 1820′ can be turned off,freeing the clones 1812 in those pens. The pens 1814′, 1820′ can beturned off simply by removing from the light pattern 216 being directedinto the housing 102 FIG. 2 the light that corresponds to pens 1814′,1820′. The now freed clones 1812 that were in pens 1814′, 1820′ can beflushed out of the chamber 110 (e.g., by flow 314) and discarded.

As shown in FIG. 17, the steps 1704 through 1710 can be repeated tocontinue growing clones 1812 in the pens 1816′, 1818′. Alternatively, atstep 1712, individual ones of the clones 1812 from pens 1814′, 1820′ canbe selected and placed as daughter clones in new pens, and the steps1704 through 1710 can be repeated to grow, test, and discard slowgrowers in the new pens. FIG. 21 shows an example in which individualdaughter clones 1812 from the pens 1816′, 1818′ in FIG. 20 are selectedand each placed in a new pen 2102. The new pens 2102 can be created andmanipulated in the same way that pens 1814, 1816, 1818, 1820 are createdand manipulated as discussed above. Individual daughter clones 1812 canbe selected and moved generally as discussed above (e.g., with lighttraps like light trap 304, 412 of FIG. 4).

FIG. 22 illustrates a process 2200 that is a variation of the process1700 of FIG. 17.

As shown in FIG. 22, one or more cells can be held in and secrete intothe pens. For example, as shown in FIG. 18C, a cell 1812 can be disposedin each of the pens 1814, 1816, 1818, 1820. Alternatively, there can bemore than one cell 1812 in each pen 1814, 1816, 1818, 1820. The cells1812 can be fused or transfected cells as discussed above with respectto FIGS. 18A-18C. Alternatively, cells 1812 can be simple cells ratherthan fused or transfected cells.

At step 2204 of FIG. 22, each pen 1814, 1816, 1818, 1820 can be examinedand the productivity of the cells 1812 in the pen can be analyzed. Forexample, one or more cells 1812 can be removed from each pen 1814, 1816,1818, 1820 and observed, tested, or the like to determine the secretionproductivity of the removed cells 1812.

At step 2206, the pens 1814, 1816, 1818, 1820 in which the cells 1812are secreting at less than a threshold level can be discarded. This canbe accomplished generally as shown in FIG. 20 and discussed above. Thatis, pens 1814, 1816, 1818, 1820 that contain low producing cells 1812can be turned off and the low performing cells 1812 washed awaygenerally in accordance with the discussion of FIG. 20 above.

Referring again to FIG. 22, the steps 2202 through 2206 can be repeatedto continue to have the cells 1812 in the remaining pens secrete, totest the secretion productivity of the cells in each pen, and discardcells 1812 in low producing pens. Alternatively, at step 2208,individual ones of the high producing cells 1812 can be selected andplaced as daughter cells in new pens (e.g., generally in accordance withthe example shown in FIG. 21), and the steps 2202 through 2206 can berepeated to have the daughter cells secrete in their new pens, test thesecretion productivity of the daughter cells in each pen, and discarddaughter cells in low secreting pens.

Although specific embodiments and applications of the invention havebeen described in this specification, these embodiments and applicationsare exemplary only, and many variations are possible.

We claim:
 1. A micro-fluidic device, comprising: a housing comprising abase and a micro-fluidic structure disposed on a surface of said base,wherein said base and said micro-fluidic structure define an interiorchamber for holding a first liquid medium and biological micro-objectssuspended therein, and wherein said micro-fluidic structure comprises:(i) a channel providing a flow path for the first liquid medium; and(ii) a plurality of physical holding pens, wherein each physical holdingpen of said plurality comprises: an enclosure; a single opening to saidchannel; and an inner wall extending from said opening into saidenclosure, said enclosure enclosing an interior space structured to holda biological micro-object suspended in a second liquid medium, and saidopening is oriented such that no part is facing directly into said flowpath of said channel, whereby, when said channel contains a flow of saidfirst liquid medium and said holding pen contains said second liquidmedium, a direct flow of said first liquid medium into said secondliquid medium in said interior space is impeded while diffusive mixingof said first liquid medium with said second liquid medium in saidinterior space is allowed.
 2. The micro-fluidic device of claim 1,wherein said inner wall of said at least one physical holding pencomprises a barrier between said opening of said at least one physicalholding pen and an inner containment space within said enclosure of saidat least one physical holding pen.
 3. The micro-fluidic device of claim1, wherein at least one of said plurality of physical holding pensfurther comprises an inner pen comprising an enclosure and an opening,wherein said inner pen is disposed inside said enclosure of said atleast one physical holding pen.
 4. The micro-fluidic device of claim 1,wherein: at least one physical holding pen of said plurality comprisesholding spaces separated by interior walls disposed inside saidenclosure of said at least one physical holding pen; and each saidholding space is configured to hold one of said biologicalmicro-objects.
 5. The micro-fluidic device of claim 1, furthercomprising: an inlet into said housing to said interior chamber; and anoutlet from said housing from said interior chamber.
 6. Themicro-fluidic device of claim 5, wherein said channel and said pluralityof physical holding pens are disposed in said interior chamber betweensaid inlet and said outlet.
 7. The microfluidic device of claim 6,wherein said plurality of physical holding pens is an array comprisingrows of physical holding pens, wherein each of said plurality ofphysical holding pens opens to said channel.
 8. The micro-fluidic deviceof claim 6, further comprising a gas exchanger disposed in said interiorchamber between said inlet and said plurality of physical holding pens.9. The micro-fluidic device of claim 1, further comprising a gasexchanger disposed in said housing.
 10. The micro-fluidic device ofclaim 1 further comprising sensors for monitoring a biological activityof said biological micro-objects in said plurality of physical holdingpens.
 11. The micro-fluidic device of claim 10, wherein said sensorsdetect the temperature, chemical composition, pH, or osmolarity of mediawithin said interior chamber.
 12. The micro-fluidic device of claim 1,wherein said micro-fluidic device comprises a plurality of saidchannels, wherein each channel of said plurality is fluidicallyconnected with a plurality of physical holding pens.
 13. Themicro-fluidic device of claim 12, wherein said plurality of channels isdisposed in said interior chamber between said inlet and said outlet.14. The micro-fluidic device of claim 13, wherein said plurality ofchannels comprises branching channels each connecting to an inputchannel and an output channel.
 15. The micro-fluidic device of claim 14,wherein each of the plurality of branching channels are connected to thesame input channel.
 16. The micro-fluidic device of claim 1, wherein allor part of said housing is gas permeable.
 17. The micro-fluidic deviceof claim 1, wherein said housing comprises a flexible material.
 18. Themicro-fluidic device of claim 1, wherein said base has a featurelesssurface.
 19. A method of processing biological micro-objects,comprising: introducing a plurality of biological micro-objects into achannel of a micro-fluidic device comprising: a housing comprising abase and a micro-fluidic structure disposed on a surface of said base,wherein said base and said micro-fluidic structure define an interiorchamber for holding a first liquid medium and biological micro-objectssuspended therein, and wherein said micro-fluidic structure comprises:(i) said channel providing a flow path for the first liquid medium; and(ii) a plurality of physical holding pens, wherein each physical holdingpen of said plurality comprises: an enclosure; a single opening to saidchannel; and an inner wall extending from said opening into saidenclosure, said enclosure enclosing an interior space structured to holda biological micro-object suspended in a second liquid medium, and saidopening is oriented such that no part is facing directly into said flowpath of said channel, whereby, when said channel contains a flow of saidfirst liquid medium and said holding pen contains said second liquidmedium, a direct flow of said first liquid medium into said secondliquid medium in said interior space is impeded while diffusive mixingof said first liquid medium with said second liquid medium in saidinterior space is allowed; moving at least one of the plurality ofbiological micro-objects into an interior space of each of the pluralityof physical holding pens of the micro-fluidic device; providing to saidchannel having said plurality of physical holding pens opening thereto aflow of a first liquid medium over a time period; and while providingsaid flow, impeding direct flow of said first liquid medium from saidflow in said channel into said interior spaces of said plurality ofphysical holding pens, wherein said interior spaces contain a secondliquid medium.
 20. The method of claim 19, wherein said impedingcomprises allowing diffusive mixing of said first liquid medium fromsaid flow with said second liquid medium in said interior spaces of saidplurality of physical holding pens.
 21. The method of claim 19, furthercomprising monitoring during said time period a characteristic of saidat least one of said plurality of biological micro-objects in saidplurality of physical holding pens.
 22. The method of claim 21, whereinsaid characteristic is a biological activity of said at least one ofsaid plurality of biological micro-objects in said plurality of physicalholding pens.
 23. The method of claim 22, wherein: said biologicalactivity comprises cloning; and said monitoring comprises monitoringsaid cloning of said at least one of said plurality of biologicalmicro-objects in said plurality of physical holding pens.
 24. The methodof claim 22, wherein: said biological activity comprises secretingsecretions; and said monitoring comprises monitoring said secretions ofsaid at least one of said plurality of biological micro-objects in saidplurality of physical holding pens.
 25. The method of claim 21, whereinsaid characteristic is a biological state of said at least one of saidplurality of biological micro-objects in said plurality of physicalholding pens.
 26. The method of claim 19, wherein said first liquidmedium is a different type of medium than said second liquid medium. 27.The method of claim 19, wherein said first liquid medium is a same typeof medium as said second liquid medium.
 28. The method of claim 19,wherein said providing comprises flowing said first liquid medium bysaid openings in said plurality of physical holding pens.
 29. The methodof claim 19, further comprising maintaining said plurality of biologicalmicro-objects in said plurality of physical holding pens during saidtime period.
 30. The method of claim 21, further comprising: selectingat least one of said plurality of biological micro-objects in saidinterior spaces of said plurality of physical holding pens; and removingsaid at least one biological micro-object from said interior space ofsaid physical holding pen.
 31. A micro-fluidic device, comprising: abase; and a micro-fluidic structure disposed on a surface of the base,the base and the micro-fluidic structure defining an interior chamberfor holding a first liquid medium, the micro-fluidic structurecomprising: (i) a channel configured to provide a flow path for thefirst liquid medium; and (ii) a plurality of physical holding pens, eachphysical holding pen comprising: an enclosure having an interior spacestructured to hold a second liquid medium; a single opening to thechannel; and an inner wall extending from the single opening into theenclosure; wherein the single opening is oriented such that no part isfacing directly into the flow path of the channel, and when the channelis configured to contain a flow of the first liquid medium and eachholding pen is configured to contain the second liquid medium, a flow ofthe first liquid medium into the second liquid medium in the interiorspace is impeded while diffusive mixing of the first liquid medium withthe second liquid medium in the interior space is allowed.